| Porcine Circovirus Disease is an infectious syndrome caused by Porcine Circovirus type 2(Porcine,circovirus,type 2,PCV2)as the main pathogen.PCV2 can damage the lymphocytes of infected pigs,cause immunosuppression and more serious harms to secondary infection and mixed infection of many pathogens.At present,there is no effective treatment for Porcine circovirus disease,and vaccination is the main means to prevent and control the disease.The detection and analysis of antibody in swine herd is an important index to evaluate the effect of vaccine immunity,virus infection and establishment of immunization program.This study was conducted to detect PCV2 antibody of 544 serum samples collected in different pig farms from Shaanxi province in 2016,And we analyzed the immune status and PCV2 infection to provided the basic data for the prevention and control of this disease.At present,the domestic use of porcine circovirus vaccine is inactivated virus vaccine and subunit vaccine,The existence of inactivated vaccine is large antigen and slow immune response;subunit vaccine has better immune effect,but it also has the problems of high cost and slow reaction..Therefore,on the basis of previous research,we cloned the Cap gene of PCV2 and the Rep gene of PCV1,and connect them through overlap PCR.Then,we successfully constructed pSCA-Cap-T2A-Rep recombinant vector as a nucleic acid vaccine to immune mice,in order to evaluate the effect of immunization,and provide new ideas and basic materials for the study of new PCV2 nucleic acid vaccine.The following results are obtained in this thesis:1.Of the 544 serum samples from Shaanxi province,PCV2 vaccine was used in 320 large-scale pig farms,and the other 224 serum samples with unclear background were detected.We can see PCV2 antibody positive serum were 368,with a positive rate of 67.6%.the positive rate of large-scale pig farms was 84.7%(271/320),and the positive rate with unclear background was 43.3%(97/224).2.PCV2 serum nucleic acids were detected by PCR of the 544 seras collected mentioned before,and we could see 103 positive nucleic acids were detected.The positive rate was 18.9%(103/544).The positive rate of the immunized pig farm was 10%(32/320),while,the pig with unclear background was 31.7%(71/224).It showed that vaccine did not prevent PCV2 infection,but could greatly reduce the infection rate;so we could see PCV2 pathogens must be detected to better prevent and control the occurrence of PCVD in addition to detect the antibody levels in the prevention and control of Porcine circovirus disease.,3.The Cap gene of PCV2 and the Rep gene of PCV1 was cloned,and was connected through overlap PCR.Finally,the pSCA-Cap-T2A-Rep recombinant vector was organised,and we transfered it into the PK15 cell lines to verify the objective gene expression by western Blot.4.After a large number of recombinant plasmids were extracted,the mice were immunized with the nucleic acid vaccines,and their immune effects were evaluated by detecting antibodies and tapping tests.The pSCA-Cap-T2A-Rep immune mice were seted up for three groups of different concentration gradient(50 μg/each body;100 μg/each body;200 μg/each body);meanwhile,another three groups with immune adjuvant were seted up the same way.Another two groups with saline were seted for control group(one with immune adjuvant,and anther with none).0 d for the first immunization,14 d for the second times,the immune dose and part the same with the first time.The blood samples were collected from the ophthalmic venous plexus at 0,14,21,and 28 Days after first immunization.The result showed: the antibody concentrations of nucleic acid immunization groups was much higher than the saline control(p<0.01);the immune adjuvant could improve the immune effect obviously(p<0.05);and the antibody level of high dose group(100 g/ only with 200 g/ only group)was significantly higher than the low dose group(50 g/ only group)(p<0.01). |
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